Cereal grains, such as corn, contain a tough outer layer of tissue called the pericarp that is primarily composed of fiber. Underneath the pericarp tissue is the nutrient-rich aleurone layer, starchy endosperm layer, and the germ (embryo), which is comparatively rich in protein and oil. In corn milling, the maize seed pericarp is approximately 5.9% of the seed by weight and contains roughly 4% protein and 0.9% oil. Since the pericarp contains the major fraction of the bran or fiber in grains, grains from which the pericarp specifically, or fiber generally, has been removed are known as “debranned” grains.
Plant pericarp tissue originates or derives from the mature ovule wall, and it constitutes the outermost tissue layer of a seed. For example, corn pericarp is the outer covering of the corn kernel. Pericarp generally comprises three distinct layers: the exocarp; the mesocarp; and the endocarp. Pericarp is produced as a byproduct of some processes. For example, corn pericarp tissue has traditionally been discarded as a byproduct of wet- and dry-milling procedures used to recover cornstarch for ethanol production. However, isolated pericarp tissue has many uses in both agricultural and industrial processes. For example, corn pericarp tissue has served as a key ingredient in the production of end products, such as corn fiber gum (an emulsifier and bulking reagent) and corn fiber oil (a nutraceutical).
The conventional separation of pericarp from other seed tissues generally proceeds by wet- or dry-milling. A wet-milling process typically begins with kernel softening in an acid steeping solution (e.g., lactic acid and sulfur dioxide) and proceeds by separation of the pericarp from the kernel (Shandera and Jackson (1986) Cereal Chem. 73:632-7) using mechanical action. The germ-free kernel is then coarsely ground into a slurry and screened through sieves to remove coarse corn fiber. The germ, which is lighter than water, is collected off of the surface of the slurry and subsequently purified to obtain corn bran containing, for example, about 91% dietary fiber. Mistry and Eckhoff (1992) Cereal Chem. 69:2296-303. In a dry-milling procedure, corn is adjusted to 20% moisture content and processed with a degerminator, so as to strip away pericarp tissue and leave intact endosperm tissue. The separated pericarp may subsequently be dried (e.g., for corn fiber oil extraction), while the starch-containing endosperm may be used, for example, in ethanol production.
Corn pericarp may also be conventionally isolated by alkali debranning and sonication. See, e.g., U.S. Pat. No. 2,472,971 (alkali debranning); Liu (2002) “Ultrasounds enhanced corn pericarp separation process,” M.S. thesis, Dept. of Food Science, University of Arkansas: Fayetteville, Ark. (sonication); Yang and Seibenmorgen (2001) “Ultrasound processing of foods—A case study of corn component separation.” ASAE Paper No. 026022, ASAE: St. Joseph, Mich. (sonication). In an alkali debranning method, the pericarp is isolated chemically by soaking in an alkaline solution (e.g., calcium hydroxide, potassium hydroxide, and sodium hydroxide) at temperatures from ambient to about 100° C. The alkali loosens the interconnecting protein matrix between the pericarp and the endosperm tissue, allowing for mechanical separation in a latter step. Sonication coupled to heat and a soaking reagent has also been shown to be effective for pericarp isolation, but the pericarp must be recovered quickly to avoid re-adhering of the protein matrix (which will occur within hours if the sonicated material is taken out of solution).
In addition to industrial uses, pericarp tissue may also be applicable in some genetic studies. The majority of genetic applications use whole seed or embryo tissue for DNA extraction. However, using such source material results in the isolation of DNA from tissues that are derived from both maternal and paternal genetic contributions. Conversely, pericarp tissue is derived from the mature ovule wall of the seed; it is derived from maternal tissue and comprises only maternal DNA. The isolation of DNA from pericarp tissue may thus be used in applications involving the analysis or use of maternal DNA, for example and without limitation, maternal lineage analysis; characterization of heterotic groups; development of phylogenetic trees; and marker assisted breeding practices (pericarp DNA genotyping applications offer the unique opportunity to evaluate the maternal genetics of a hybrid plant line). Identifying maternally-inherited genes and maternally-inherited chromatin may allow breeders to select progeny that are genetically similar (or dissimilar) to one parent or the other for reciprocal recurrent selection and test-crossing.
A higher degree of pericarp tissue purity is required for the use of this maternally-derived tissue in genetics, compared to its use in other applications. Thus, methods for isolating pericarp tissue for non-genetic uses may not be appropriate or adaptable to isolate pericarp for genetic applications. Contaminating endosperm, aleurone, and germ tissue must be removed from pericarp prior to DNA extraction, as even the smallest amount of contaminating DNA in a sample of pericarp DNA may render the sample useless or lead to erroneous or uninterpretable results. For at least this reason, plant geneticists are unlikely to accept a priori that a pericarp isolation method will be adequate for their purposes, absent some confirmation that the isolated pericarp sample is not contaminated by endosperm, aleurone, or germ tissue. In addition to the absence of other tissue from the pericarp, the purity of isolated pericarp DNA should be in the range of 1.8-2.0 (as measured by the A260/A280 absorption ratio) to ensure adequate results in downstream DNA analysis.